不同浓度哇巴因对人血管内皮细胞的作用

Effect of ouabain at different concentrations on human vascular endothelial cells

目的研究不同浓度哇巴因对血管内皮细胞(HUVEC)的作用及生理浓度哇巴因激活的血管内皮细胞早期反应基因。方法MTT法观察哇巴因对血管内皮细胞增殖的影响,台盼蓝染色及乳酸脱氢酶活性测定细胞活力。免疫组织化学染色法测定细胞核增殖抗原(PCNA)表达情况。以包含8464条人类基因的DNA芯片检测血管内皮细胞受生理浓度哇巴因活化后的基因表达谱,并从中筛选出早期反应基因。结果生理浓度(0.3-0.9nmol/L)的哇巴因能刺激细胞增殖,且这种增殖不与剂量呈正相关,刺激增殖的最适浓度为0.3nmol/L,最大效应作用时间为1-2h。0.9-1.8nmol/L的哇巴因抑制内皮细胞的增殖,引起细胞水肿和凋亡。但10nmol/L的高浓度哇巴因却能够引起细胞的增殖。血管内皮细胞受哇巴因作用2h后,基因表达谱研究显示340条基因出现表达差异,其中上调的共有145条,多数与细胞代谢和转录调控相关,显著上调的6条。结论哇巴因在参与维持血管内皮细胞的正常增殖及高血压引起的血管重塑中起重要作用。

AIM: To study the effect of ouabain at different concentrations on human vascular endothelial cells (HUVEC), and early endothelial responses to ouabain in physiological concentration by genomic-scale gene expression analysis. METHODS: The proliferation of HUVEC was determined by MTT method. The vitality of cells was determined by LDH, and PCNA was assessed by using immunohistochemistry. The response of endothelial cells to ouabain was explored with a complementary DNA microarray representing ~8 464 different human genes. RESULTS: Ouabain stimulated the proliferation of HUVEC at physiological concentration (0.3-0.9 nmol/L). Ouabain at pathological concentration (0.9-1.8 nmol/L) inhibited proliferation and induced cellular swelling and apoptosis. However, the effect of ouabain at the concentration of 10 nmol/L also stimulated proliferation. The results of mRNA profiles analysis indicated that 340 of genes were differentially expressed, among them 145 were upregulated and 6 were upregulated significantly. The upregulated genes were mainly related to cellular metabolism and gene transcription. CONCLUSIONS: Ouabain has important role in controlling the growth and metabolism of vascular endothelial cell and vascular remodeling in the pathogenesis of hypertension.